Cesium fluoroaluminate

Endpoint summary

Key value for chemical safety assessment

Effects on fertility

Description of key information

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422 (2016); GLP) with cesium fluoroaluminate was conducted in rats. Fertility of male and female animals was unaffected during the course of this study. Adverse effects were observed for gestation length, gestation index, number of implantation sites, number of resorptions and stillbirths at the highest dose level (750 mg/kg bw/day). However, the adverse findings are attributed to the overt toxicity with mortality (6 out of 10 females died) at the highest dose level and should therefore not be considered as primary reprotoxic effect. Therefore, cesium fluoroaluminate is not considered to be a primary reprotoxic substance, since adverse effects on the fertility were only seen at signs of excessive toxicity, involving death of 6 of 10 females and 2 of 10 males. Consequently, the criterial for classification and labelling as reprotoxic (impairment of fertility) are not met.

Link to relevant study records

Reference

Endpoint:

reproductive toxicity, other

Remarks:

Combined repeated dose toxicity study with the reproductive/developmental toxicity screening test

Type of information:

experimental study

Adequacy of study:

key study

Study period:

2018-03-21 to 2018-06-05

Reliability:

1 (reliable without restriction)

Rationale for reliability incl. deficiencies:

guideline study

Reason / purpose for cross-reference:

reference to same study

Qualifier:

according to guideline

Guideline:

OECD Guideline 422 (Combined Repeated Dose Toxicity Study with the Reproduction / Developmental Toxicity Screening Test)

Version / remarks:

2016-07-29

Deviations:

no

GLP compliance:

yes (incl. QA statement)

Remarks:

signed 2017-05-08

Limit test:

no

Specific details on test material used for the study:

STABILITY AND STORAGE CONDITIONS OF TEST MATERIAL
- Storage condition of test material: at +10 °C to +25°C in a tightly closed original container, stored in a dry, cool and well venitlated place.

Species:

rat

Strain:

other: Crl:CD(SD)

Details on species / strain selection:

The rat is a commonly used rodent species for such studies.

Sex:

male/female

Details on test animals or test system and environmental conditions:

TEST ANIMALS
- Source: Charles River Laboratories, Research Models and Services, Germany GmbH, Sandhofer Weg 7, 97633 Sulzfeld, Germany
- Females nulliparous and non-pregnant: yes
- Age at first administration: males: 74 days; females: 75 days
- Weight at first administration: males: 407.0 g - 477.5 g; females: 242.5 g - 278.0 g
- Housing (with the exception of the mating period): kept singly in MAKROLON cages (type III plus) with a basal surface of approx. 39 cm x 23 cm and a height of approx. 18 cm; bedding material: granulated textured wood (Granulat A2, J. Brandenburg)
- Diet (ad libitum): certified commercial diet (ssniff® R/Z V1324, ssniff Spezialdiäten GmbH); food residue was removed and weighed.
- Water (ad libitum): tap water
- Acclimation period: 7 days

ENVIRONMENTAL CONDITIONS
- Temperature: 22 °C ± 3 °C (maximum range)
- Relative humidity:55 % ± 15 % (maximum range)
- Air changes: 15 to 20 air changes/hour
- Photoperiod (hrs dark / hrs light): 12/12

Route of administration:

oral: gavage

Vehicle:

water

Details on exposure:

- PREPARATION OF DOSING SOLUTIONS:
The test item was suspended in the vehicle to provide dose concentrations of 37.5, 125 or 375/250 mg/mL
The test item formulations were freshly prepared every day and were adjusted to the animal's actual body weight daily.
The control animals received the vehicle at the same administration volume daily in the same way.

- Administration volume: 2 mL/kg bw/day

Details on mating procedure:

- M/F ratio per cage: 1 male / 1 female
- Length of cohabitation: the female was placed with the same male until pregnancy had occurred or 2 weeks had elapsed.
- Proof of pregnancy: vaginal plug / sperm in vaginal smear referred to as day 0 of gestation.

Analytical verification of doses or concentrations:

yes

Details on analytical verification of doses or concentrations:

For the analysis of the test item-vehicle formulations, samples of approx. 15 mL were taken at the following times and stored at ≤-20°C ± 10% until analysis:
1) At start of the treatment period (first dosing day):
- analysis of stability and concentration: immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature (3 samples / dose level group).
- homogeneity: at the start of administration, during (middle) administration and before administration to the last animal/dose level group (3 samples / dose level group plus one sample/control).

2) At dose change (highest dose group, males)
- analysis of stability and concentration: immediately after preparation of the test item-vehicle formulation as well as after 8 and 24 hours storage at room temperature (3 samples of highest dose group).
- homogeneity: at start of administration, during (middle) administration and before administration to the last animal/dose level group (3 samples of highest dose group).

3) towards the end of the treatment period (when the majority of animals was dosed):
- analysis of concentration: during treatment always before administration to the last animal/dose level group (1 sample / group; low and intermediate dose groups; 2 samples from highest dose group: 1 sample from the male and 1 sample from the female group).

Method:
The quantification of the test item cesium fluoroaluminate in the test item-vehicle formulations was performed by the determination of the aluminium and the cesium content. For determination of aluminium, all samples and controls were analysed by inductively coupled plasma optical emission spectrometry (ICP-OES) (three determinations) after dilution with pure water (only for test item containing samples) and (further) with nitric acid containing water (samples and controls). For determination of cesium, all samples and controls were analysed by flame atomic absorption spectrometry (FAAS) (three determinations) after dilution with pure water (only for test item containing samples) and (further) dilution with hydrochloric acid containing water and addition of KCl-solution (samples and controls).

The methods were validated for selectivity, linearity, limit of quantification (LOQ), precision (reproducibility) and accuracy (recovery). The validity of the applied methods has been proven.

Results:
These results indicated correctly prepared and homogenised test item vehicle mixtures.

Aluminium content:
Mean recovery of the samples was 98 % with a relative standard deviation of 5 %. The maximum was found at 103% and the minimum at 80 %.

Range of % nominal concentration:
- stability and concentration (immediately after preparation on test day 15 (first
day of administration) as well as after 8 and 24 hours storage at room temperature): 79.97 % - 100.37 %
- stability and concentration (on test day 38 (dose change for high dose level, males), immediately after preparation as well as after 8 and 24 hours storage
at room temperature): 100.63 % - 102.00 %
- stability and concentration (on test day 48 (towards the end of the treatment
period), before administration to the last animal): 93.85 % - 102.92 %
- homogeneity (on test day 15, at the start of administration, during administration and before administration to the last animal): 92.55 % - 102.36 %
- homogeneity (on test day 38 (dose change for high dose level, males), at the start of administration, during administration and before administration to the last animal): 97.70 % - 99.53 %

Cesium content:
Mean recovery of the samples was 101 % with a relative standard deviation of 4 %. The maximum was found at 109% and the minimum at 89 %.

Range of % nominal concentration:
- stability and concentration (immediately after preparation on test day 15 (first
day of administration) as well as after 8 and 24 hours storage at room temperature): 94.72 % - 109.34 %
- stability and concentration (on test day 38 (dose change for high dose level, males), immediately after preparation as well as after 8 and 24 hours storage
at room temperature): 97.82 % - 101.07 %
- stability and concentration (on test day 48 (towards the end of the treatment
period), before administration to the last animal): 99.19 % - 102.60 %
- homogeneity (on test day 15, at the start of administration, during administration and before administration to the last animal): 88.76 % - 106.30 %
- homogeneity (on test day 38 (dose change for high dose level, males), at the start of administration, during administration and before administration to the last animal): 97.61 % - 104.12 %.

Duration of treatment / exposure:

- males: 35 treatment day (2 weeks prior to mating (from test day 15 until test day 29), during the mating period (14 days at maximum) and during the post-mating period until one day before sacrifice)
- females: 50 to 62 treatment days (2 weeks prior to mating, during the mating period (14 days at maximum) and during the lactation period until lactation day 13).

Frequency of treatment:

once daily

Dose / conc.:

75 mg/kg bw/day (actual dose received)

Remarks:

administered to both sexes

Dose / conc.:

250 mg/kg bw/day (actual dose received)

Remarks:

administered to both sexes

Dose / conc.:

750 mg/kg bw/day (actual dose received)

Remarks:

administered to both sexes, but due to the premature death of 2/10 males, the dose level was reduced from 750 to 500 mg/kg bw/day on test day 38 for the males.

Dose / conc.:

500 mg/kg bw/day (actual dose received)

Remarks:

starting on test day 38, this dose was administered to males instead of the 750 mg/kg bw/day dose due to premature deaths of the males in the high dose group.

No. of animals per sex per dose:

10 males / 10 females

Control animals:

yes, concurrent vehicle

Details on study design:

- Dose selection rationale: the dose levels were selected based on available toxicological data and a 21-day dose range finding study (please refer to section 7.8.1 Toxicity to reproduction: s_Hansen_2018_DRF). In the 21-day dose range finding study, cesium tetrafluoroaluminate was administered orally to male and female rats at dose levels of 2, 10, 50, 100, 200, 600 or 1000 mg/kg bw/day for 3 weeks from test days 1 to 21.
At 1000 mg/kg bw/day one of 5 male animals died prematurely (found dead on the day of necropsy on test day 22) after 21 days of dosing. A post-dosing salivation (salivation started immediately to 5 minutes after dosing and disappeared again between 20 and 60 minutes after dosing) was noted from a dose level of 50 mg/kg bw/day onwards. The observation of salivation was related to the dose level (1 of 5 affected male animal at a dose level of 50 mg/kg bw/day and 5 of 5 affected male and female animals from 200 mg/kg bw/day onwards).
A reduced body weight and body weight gain was noted for the male animals at 1000 mg/kg bw/day but not for the female animals.
After 21 days of dosing statistically significant changes were noted for one or both sexes for several haematological parameters at 100, 200, 600 and 1000 mg/kg bw/day. In detail, slightly to moderately decreased levels were noted for the haemoglobin concentration, the number of red blood cells, the haematocrit value and the mean corpuscular volume.
A moderate to marked increase was noted for the number of white blood cells at dose levels of 600 and 1000 mg/kg bw/day.
The macroscopic examination at necropsy after 21 days of dosing revealed test item-related changes at 1000 mg/kg bw/day for the male animals in the form of small testes (1 of 5 males) and stomach changes (thickened mucosa, ulcera; 3 of 5 males).

Positive control:

none

Parental animals: Observations and examinations:

CAGE SIDE OBSERVATIONS: Yes
- Time schedule:
clinical signs: before and after dosing at each time of dosing as well as regularly throughout the working day from 7:00 a.m. to 3:45 p.m. On Saturdays and Sundays animals were checked regularly from 7:00 a.m. to 11:00 a.m. with a final check performed at approx. 3:30 p.m.
mortality: early in the morning and again in the afternoon of each working day as well as on Saturdays and Sundays, a similar procedure was followed with a final check at approx. 3.30 p.m.
- Cage side observations checked: behavioural changes, signs of difficult or prolonged parturition, and all signs of toxicity as well as mortality

DETAILED CLINICAL OBSERVATIONS: Yes
- Time schedule: once before the first exposure (to allow for within-subject comparisons) and once a week thereafter (performed at least 2 hours after dosing)

BODY WEIGHT: Yes
- Time schedule for examinations:
1) Males: weekly starting during the premating period and at sacrifice

2) Females:
premating period: weekly
gestation period: gestation days 0, 7, 14 and 20
lactation period: lactation days 1,4 and 13

FOOD CONSUMPTION AND COMPOUND INTAKE: Yes
- Food intake per rat (g/rat/week) was calculated using the total amount of food given to and left by each rat in each group upon completion of a treatment week (pre-mating and gestation) or treatment period (lactation). From these data the relative food consumption (in g/kg bw/day) was determined using the following
formula: Relative food consumption = (total food given (g) - total food left (g))/number of animal days* x body weight (kg)
*the term 'animal days' counts one animal day for each animal alive for a whole day; it is assumed that on the day of death an animal does not eat.

Food residue (or total food left) was weighed and recorded as followed:
- Pre-mating period
males and females: TD8 and TD15
- Mating period:
males and females: none
- Gestation period
males: not applicable
females: GD7, GD14 and GD21
- Lactation period
males: not applicable
females: LD1, LD8 and LD12

FOOD EFFICIENCY:
- Body weight gain in kg/food consumption in kg per unit time X 100 calculated as time-weighted averages from the consumption and body weight gain data: No

WATER CONSUMPTION AND COMPOUND INTAKE: Yes
- Time schedule for examinations: daily by visual appraisal throughout the study

OPHTHALMOSCOPIC EXAMINATION: No

HAEMATOLOGY: Yes
- Time schedule for collection of blood:
at the end of the premating period on test day 29
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- Animals fasted: Yes, overnight
- How many animals:
5 animals/sex/group
- Parameters checked:
haemoglobin content, erythrocytes, leucocytes, differential blood count (relative and absolute; neutrophilic, eosinophilic and basophilic granulocytes, lymphocytes and monocytes as well as large unstained cells), reticulocytes, platelets, haematocrit value, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, thromboplastin time and activated partial thromboplastin time

CLINICAL CHEMISTRY: Yes
- Time schedule for collection of blood:
at the end of the premating period on test day 29
- Animals fasted: Yes, overnight
- How many animals:
5 animals/sex/group
- Parameters checked:
albumin, globulin, albumin/globulin ratio, bile acids, bilirubin (total), cholesterol (total), creatinine, glucose, protein (total), blood urea, calcium, chloride, potassium, sodium, sodium/potassium ratio, blood urea/creatinine ratio, lactate dehydrogenase, alanine aminotransferase, alkaline phosphatase and aspartate aminotransferase

URINALYSIS: No

NEUROBEHAVIOURAL EXAMINATION: Yes
- Time schedule for examinations:
at least two hours after dosing and before any blood sampling for laboratory examinations:
males: test day 48
females: test day 64, 65, 66, 70 or 73
- Dose groups that were examined:
all dose groupy (5 animals/sex/dose)
- Battery of functions tested: sensory activity / grip strength / motor activity

IMMUNOLOGY: No

THYROID HORMONE (T4) DETERMINATION: Yes
- Time schedule for examinations: at scheduled sacrifice (always at the same time in the morning)
- Animals fasted: Yes, overnight
- Anaesthetic used for blood collection: Yes, isoflurane anaesthesia
- How many animals: all parental males

FURTHER OBSERVATIONS:
- duration of pre-coital time
- gestation length
- number of implantation sites

Oestrous cyclicity (parental animals):

During the 14-day pre-exposure period (TD 1 to TD 14), the oestrus cycle of the female animals was monitored to yield study groups of at least 10 animals each with a normal oestrus cycle. Animals that failed to exhibit typical 4 to 5 day cycles were excluded from the randomization process on test day 14 that was performed to allocate the female animals to the test groups of the main study. Most of the female animals that were used for the main study revealed 2 or 3 typical 4 to
5 day cycles during the 14-day pre-exposure period.
After the allocation of the animals to the study groups and the start of treatment, the oestrus cycle was further monitored daily during the pre-mating period and the
mating period until one day before a positive mating sign was noted.
Finally, a vaginal smear was taken in the morning of the day of scheduled necropsy to determine the stage of the oestrus cycle and allow correlation with the histopathology of the female reproductive organs.

Sperm parameters (parental animals):

Parameters examined in P male parental generations: testis weight, epididymis weight, qualitative stages of spermatogenesis and testicular structure

Litter observations:

STANDARDISATION OF LITTERS
- Performed on day 4 postpartum: yes
- If yes, the litters were adjusted to 10 pups/litter (5 males / 5 females pups, if enough pups were available for each sex) by eliminating (culling) surplus pups.

PARAMETERS EXAMINED
The following parameters were examined in F1 offspring as soon as possible after delivery:
number and sex of pups, stillbirths, live births, runts, presence of gross anomalies and behavioural abnormalities
Live pups were counted, sexed and weighed on post-natal days 1, 4 and13.
On post-natal day 4 before litter adjustment the ano-genital distance of all pups was determined usng a scale.
Nipples were counted in all male pups on post-natal day 13 (shortly before scheduled sacrifice).
Clinical signs were observed daily.

THYROID HORMONE (T4) DETERMINATION
- Time schedule for examinations: postnatal day 13
- Animals fasted: No
- How many animals: at least 2 pups/litter (if possible from one male and one female; all litters

Postmortem examinations (parental animals):

GROSS PATHOLOGY:
The animals were sacrificed at the following times:
- males: on test day 50
- dams (surviving dams): on lactation day 14
At the time of sacrifice or premature death during the study, the adult animals were examined macroscopically for any abnormalities or pathological changes. Special attention was paid to the organs of the reproductive system.
During necropsy the number of implantation sites was recorded in the female animals.
Apparently non-pregnant uteri were placed in a 10% aqueous solution of ammonium sulfide for about 10 minutes to stain possible implantation sites in the endometrium according to SALEWSKI.
All superficial tissues were examined visually and by palpation and the cranial roof removed to allow observation of the brain, pituitary gland and cranial nerves. After ventral midline incision and skin reflection all subcutaneous tissues were examined. The condition to the thoracic viscera was noted with due attention to the thymus, lymph nodes and heart.
The abdominal viscera were examined before and after removal; the urinary bladder was examined externally and by palpation. The gastro-intestinal tract was examined as a whole and the stomach and the caecum were incised and examined. The lungs were removed and all pleural surfaces were examined under suitable illumination.
The liver and the kidneys were examined. Any abnormalities in the appearance and size of the gonads, adrenals, uterus, intra-abdominal lymph nodes and accessory reproductive organs were recorded.

ORGAN WEIGHTS.
The weight of the following organs of all adult male and female animals was determined before fixation (where applicable): adrenal glands, brain, epididymis, heart, kidneys, liver, ovaries, spleen, testicles, thyroid, thymus, prostate, seminal vesicles with coagulating glands, uterus including cervix

HISTOPATHOLOGY:
The following organ(s) or parts thereof of all adult male and female animals were fixed in modified Davidson's solution or 7% buffered formalin: epididymis, testicles, gross lesions observed, ovaries and oviducts, prostate, seminal vesicles with coagulating glands, thyroid (including parathyroids), uterus (including cervix) and vagina.
Any other organs displaying macroscopic changes were also preserved.

5 male and 5 female animals from each group were selected for histopathology examination (prematurely deceased animals were additionally examined) and the following organs from the selected animals and from every deceased or prematurely sacrificed animal were fixed for histopathology examination: eye with optic nerve (2), epididymis (2), testicle (2), adrenal gland (2), bone, bone marrow (os femoris), brain (cerebrum, cerebellum, brain stem (pons)), gross lesions observed, heart (3 levels: right and left ventricle, septum), intestine, small (duodenum, jejunum, ileum, incl. Peyer's patches, Swiss roll method), intestine, large (colon, rectum), kidney and ureter (2), liver, lungs (with mainstem bronchi and bronchioles), preserved by inflation with fixative and then immersion, lymph node (1, cervical), lymph node (1, mesenteric), mammary gland, muscle (skeletal), nerve (sciatic), oesophagus, ovary and oviduct, pituitary, prostate and seminal vesicles with coagulating glands, spinal cord (3 sections), spleen, stomach, thyroid (including parathyroids), thymus, tissue masses or tumors including regional lymph nodes), tongue (including base), trachea (including larynx), urinary bladder, uterus (including cervix) and vagina.

Full histopathology was performed on the preserved organs of the selected parental animals of the control group and the highest dose group. The organs listed above were examined histologically after preparation of paraffin sections and haematoxylin-eosin staining. Parathyroids cannot always be identified macroscopically. They were examined microscopically if in the plane of section and in all cases where they are noted were grossly enlarged.
In addition, frozen sections of the heart, liver and one kidney were prepared, stained with Oil Red O and examined histologically.
Detailed histopathologic examination was performed on one testicle and one epididymis (with special emphasis on the qualitative stages of spermatogenesis and histopathology or interstitial testicular structure) of the selected males of the control group and the highest dose group following H-E and PAS staining.

In addition, haematoxylin-eosin stained paraffin sections of the following organs of the low and mid dose group animals were prepared and evaluated: adrenal glands, heart, kidneys, lungs, lymph node (mesenteric), lymph node (cervical), skeletal miscle, spleen, stomach, testicle, thymus, urinary bladder, uterus and vagina.

H-E stained sections of both testicles of three further high dose males were prepared and examined to clarify macroscopic findings in these animals.

Postmortem examinations (offspring):

SACRIFICE
The pups were sacrificed on postnatal day 13.

GROSS NECROPSY
Dead pups and pups sacrificed at day 13 post-partum were examined externally for gross abnormalities. The external reproductive genitals were examined for signs of altered development.

HISTOPATHOLOGY / ORGAN WEIGTHS
The thyroid of 1 male and 1 female pup from each litter was fixed in 7% formalin.
Thyroid weight was determined after fixation.
The thyroid of selected pups was histopatholoically examined.

Statistics:

Homogeneity of variances and normality of distribution were tested using the BARTLETT’s and SHAPIRO-WILK’s test. In case of heterogeneity and/or nonnormality of distribution, stepwise transformation of the values into logarithmic or rank values was performed prior to ANOVA. If the ANOVA yielded a significant
effect (p ≤ 0.05), intergroup comparisons with the control group were made by the DUNNETT’s test (p ≤ 0.01 and p ≤ 0.05).
The statistical evaluation of non-parametrical values was done by comparison of the group values using the FISHER or the Chi² test.

Reproductive indices:

- female fertility index [%] = (number of pregnant rats / number of rats used) x 100
The female fertility index reflects the total number of dams that had achieved pregnancy, including dams which delivered at term, aborted or had fully resorbed
litters.
- gestation Index [%] = (number of dams with live pups / number of pregnant rats) x 100
- birth Index [%] = (total number of pups born (alive + dead) / number of implantation scars) x 100
- post-implantation loss [%] = (implantations - living fetuses / implantations) x 100

Offspring viability indices:

- live birth index [%] = (number of pups alive on day 0/1 of lactation / total number of pups (alive + dead)) x 100
- viability index [%] (pre-cull) = (number of pups alive on day 4 (pre cull) / number of pups alive on day 0/1) x 100
- viability index [%] (post cull) = (number of pups alive on day 13 / number of pups alive on day 4 (post cull)) x 100

Clinical signs:

effects observed, treatment-related

Description (incidence and severity):

Clinical signs:
1) Males:
- 750/500 mg/kg bw/day: a haemorrhagic nose/snout for 3/8 surviving animals was observed on several test days (considered to be test item-related).

2) Females:
-750 mg/kg bw/day: piloerection was noted for all 4 surviving females during the lactation period on altogether 36 test days (considered to be test item-related).
- premature deaths (750 mg/kg bw/day): the observation of piloerection that was noted for 5 of the 6 prematurely deceased animals was also noted for all 4 surviving animals and therefore considered to be test item-related and not as a pre-mortal symptom

Dermal irritation (if dermal study):

not examined

Mortality:

mortality observed, treatment-related

Description (incidence):

1) Males:
750/500 mg/kg bw/day: 2/10 male animals died prematurely on test days 30 and 36, which was considered to be test item-related due to changes in the laboratory parameters, reductions in body weight and changes in organ weight.

2) Females:
750 mg/kg bw/day: 6/10 females died during the lactation period between test days 54 to 64 (corresponding to lactation days 1 to 9). Due to the high incidence of prematurely deceased high dosed females, these premature deaths were considered to be test item-related.

Body weight and weight changes:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 750/500 mg/kg bw/day: a statistically significantly reduced body weight in comparison to the control group was noted from test day 29 until sacrifice on test day 50 (considered to be test item-related). In detail, on test day 29 the body weight of the males was 7.7 % below the value of the control group (p ≤ 0.05). Thereafter the difference in body weight slightly increased. On test day 49 (last day of dosing) the body weight of the animals was 9.5% below the value of the control group (p ≤ 0.01) and at sacrifice (test day 50) the body weight of the male animals was 11.0 % below the control value (p ≤ 0.01). The percentage of body weight gain was decreased in the high dose group in comparison to the control group and the 75 and the 250 mg/kg bw/day dose groups.
The body weight at autopsy for the 8 surviving male animals of the 750/500 mg test item/kg bw/day dose group was 10.7 % below the value of the control group (p ≤ 0.01). This was considered to be test item-related and confirmed the live body weight on test day 50 (11.0 % below the control).
- premature death (750/500 mg/kg bw/day): one male showed a marked reduction in body weight before death (minus 30.6 % between test day 15 and test day 29

2) Females:
- 750 mg/kg bw/day: a statistically significantly (p ≤ 0.05 or 0.01) reduced body weight was noted from gestation day 14 onwards until the end of the lactation period. This reduction in body weight in comparison to the control group was considered to be test item-related. In detail, the mean body weight of the females was 7.5 % below the control value on gestation day 14 (p ≤ 0.05). At the beginning of the lactation period (lactation day 1) the mean body weight of the 9 living females (one female died during littering on lactation day 1) was 14.4 % below the control value (p ≤ 0.01). During the further course of the lactation period 5 further females of the treatment group died and the mean body weight of the 4 surviving animals on lactation day 13 was 21.9 % below the value of the control group (p ≤ 0.01). In accordance with the reduced body weights that were noted during the gestation and the lactation period, body weight gain for these periods was reduced at the high dose level in comparison to the control group.
A slight and statistically not significant reduction in body weight at autopsy (9.6% below the value of the control group) was noted for the 4 surviving female animals on lactation day 14. This was considered to be test item-related. Hence, the reduction in the body weight at autopsy was in agreement with the test item-related reduction in live body weight that was noted on lactation day 13 (21.9 % below the control value).

Please refer to section "Overall remarks, attachments".

Food consumption and compound intake (if feeding study):

effects observed, treatment-related

Description (incidence and severity):

1) Females:
- 750 mg/kg bw/day: during the second week (lactation days 8 to 12) of the lactation period a reduction in food consumption (54.0% below the value of the control group, p ≤ 0.01) was noted for the 4 surviving female animals of the treatment group. This may be caused by the damaged teeth that were noted for the 4 surviving females at necropsy and/or the poor health condition of these 4 animals in general, as demonstrated by the reduced body weight or the poor results of the neurological screening. Due to these reasons, the reduced food consumption of the 4 surviving animals during the second week of the lactation period is considered to be test item-related.

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

no effects observed

Ophthalmological findings:

not examined

Haematological findings:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
Note: The 5 selected males of the 750/500 mg/kg bw/day dose group that were selected for the laboratory examinations included the 2 prematurely deceased animals).
- 250 mg/kg bw/day: a slight and statistically not significant increase was noted for the number of white blood cells (47.5 % above the value of the control group). Regarding the subunits of the white blood cells, statistically significant increases were noted for the numbers of neutrophilic and eosinophilic granulocytes (p ≤ 0.01 and p ≤ 0.05, respectively). The increases in the number of neutrophilic and eosinophilic granulocytes were considered to be test item-related, as more pronounced increases were noted at the750/500 mg/kg bw/day dose level (dose-response relationship). Furthermore, in case of the neutrophilic granulocytes 3 of 5 individual
values from the males of the 250 mg/kg bw/day dose group were in the upper range of the
laboratory background data, another 2 of 5 values slightly above the upper range. In case of the eosinophilic granulocytes 4 of 5 values were slightly above the upper value of the laboratory background data.

- 750/500 mg/kg bw/day: the increase in the number of white blood cells was more pronounced as at the 250 mg/kg bw/day dose level and statistically significant (143.3 % above the value of the control group; p ≤ 0.05). The cell numbers of nearly all subunits of the white blood cells (cell numbers of lymphocytes, monocytes, neutrophilic, eosinophilic and basophilic granulocytes, with the exception of the small population of large unstained cells) were markedly increased (statistically significant or not; p ≤ 0.01 or 0.05)). All changes that were noted at the 750/500 mg/kg bw/day dose level for the above mentioned parameters were considered to be test item-related. However, it has to be mentioned that a high variability was noted for the above mentioned parameters at the 750/500 mg test item/kg bw/day) dose level. Markedly increased numbers of white blood cells, lymphocytes, monocytes, neutropholic, eosinophilic and basophilic granulocytes were especially noted for the 2 prematurely deceased animals (one animal died 1 day after blood withdrawal on test day 30 and one animal died 7 days after blood withdrawal on test day 36) and one surviving animal. In contrast, the values of the above mentioned parameters for other two surviving animals were in the range of the control group (only for the number of neutrophilic granulocytes an increased value was noted for one animal).

For white blood cells, lymphocytes, monocytes,
neutrophilic, eosinophilic and basophilic granulocytes, the individual values from three high
dosed males were nearly all above the laboratory background range (the only exception was the number of eosinophilic granulocytes for one male). The values for the remaining 2 high dosed males were above or within the range of the laboratory background data.

2) Females:
Note: The 5 females of the 750 mg/kg bw/day dose level that were selected for the laboratory examinations included the 4 surviving females and one prematurely deceased female.
- 250 and 750 mg/kg bw/day: increased numbers of neutrophilic granulocytes, eosinophilic granulocytes and monocytes were noted at the intermediate and the high dose level (250 or 750 mg test item/kg bw/day) (p ≤0.05 / 0.01 or not statistically significant), which were considered
to be test item-related.

In case of the number of neutrophilic granulocytes a clear dose response-relationship was noted for the increase of the number of neutrophilic granulocytes from the intermediate dose group to the high dose group (from 62.2 % to 98.3 % above the control value). Furthermore, at the intermediate dose group 2 of 5 individual values and at the
high dose level 4 of 5 individual values were above the laboratory background range.

A dose response-relationship was also noted for the increase in the number of monocytes from the intermediate dose group to the high dose group (from 65.0 % to 80.0 % above the control value). With the exception of the value from one animal (high dose group animals), all individual values from the animals of the intermediate and the high dose group were above the individual values from the control group and in the upper range of the laboratory background data.

In case of the number of eosinophilic granulocytes all individual values from the females of the intermediate dose group (5 of 5) and 3 of 5 individual values from the females of the high dose group were above the values from the control group and above the laboratory background range. Though a dose response-relationship was missing, the distinctly increased numbers of eosinophilic granulocytes in comparison to the control
group that were noted for altogether 8 of 10 females of the intermediate and the high
dose group were considered to be test item-related.

Please refer to section "Overall remarks, attachments".

Clinical biochemistry findings:

effects observed, treatment-related

Description (incidence and severity):

1) Males:
- 250 mg/kg bw/day: a slight but statistically significantly decreased potassium concentration was noted (p ≤ 0.01). Though all individual values were clearly within the laboratory background data, they were all slightly below the individual values of the control group. This small reduction was considered to be test item-related due to the test item-related reductions that were noted for the potassium concentrations at the 750/500 mg/kg bw/day dose levels of the male and more pronounced of the female animals.

- 750/500 mg/kg bw/day: test item-related changes were noted for the potassium concentration, the aspartate aminotransferase activity, the protein (total), the albumin and globulin concentration and the urea in blood concentration. The findings are described in detail below.
A statistically significantly decrease in potassium concentration was noted in males (p ≤ 0.01). A marked decrease in the potassium concentration was noted for one surviving male animal and one prematurely deceased animal (values below or at the lower range of laboratory background data), whereas the potassium concentrations of the remaining 3 animals remained at the control level. Though only 2 of 5 animals showed a decreased potassium concentration at the high dose group, this was considered to be test item-related, as a decreased potassium level was also noted for the female animals of the high dose group.

Due to the test item-related decrease in the potassium concentration, a test item-related
increase was noted for the sodium / potassium ratio.

Furthermore, a statistically significantly increase in aspartate aminotransferase activity was observed at the high dose group (p ≤ 0.01). An increased aspartate aminotransferase activity that was above the upper range of the background data was noted for 4 of the 5 examined male animals.

A slight but statistically significant decrease was noted at the high dose level for the protein (total) concentration. In detail, for the protein (total) concentration, 4 of 5 individual values of the high dose group were below the lowest value of the control group and 2 values were slightly below the lower value of the laboratory background data.
Furthermore, there is a tendency for a dose-response relationship in the decrease of the
protein (total) concentrations from the intermediate dose group to the high dose group. Due to the observations listed above, the decreased protein (total) concentration that was noted at the high dose level was considered to be test item-related.

The decreased protein (total) concentration was due to decreased albumin and globulin concentrations at the high dose level (p ≤ 0.05 or p ≤ 0.01). As both parameters were decreased in a dose-dependent manner, the decreased albumin and globulin concentrations that were noted at the high dose level were considered to be test item-related. This assessment was made for the albumin and the globulin concentration,
though in case of the globulin concentrations, all individual values of the high dose group
were within the range of the laboratory background data.

A moderately but statistically not significant increase was noted for the urea in blood
concentration. Though this increase was mainly due to a markedly increased level of the one
prematurely deceased animal, it was considered to be test item-related as the
urea in blood concentration of the high dosed females was also increased.

2) Females:
- 750 mg/kg bw/day: test item-related changes were noted for the electrolyte levels (potassium, chloride and calcium concentration), the urea in blood concentration, the cholesterol concentration and the aspartate aminotransferase activity.

A statistically significantly decrease was noted for the potassium and chloride concentration and a statistically significantly increase for the calcium concentration (p ≤ 0.05 or p ≤ 0.01).

The decrease of the potassium concentration was highly significant. All individual values were markedly below the lowest value of the other test groups (control, 75 and 250 mg/kg bw/day group) and at the lower range of the laboratory background data.

A clear effect was also noted for the chloride concentration. Though the difference was rather small (3.3 % below the control value; p ≤ 0.01), 4 of 5 individual values of the high dose group were below the lower value of the control group (only the value of one 750 mg/kg bw/day dose group female was at the same level as the lowest value of the control group). Furthermore, all values were below or at the lower range of background data.

For the calcium concentration, 3 of 5 individual values from the high dose group were above or at the upper range of the background data.

No test item-related influence was noted on the sodium concentration. A slightly reduced sodium concentration was noted at the low and the high dose level. However, as no dose response relationship was noted, these reductions were considered to be spontaneous. Due to the test item-related decrease of the potassium concentration at the high dose level, a test item-related increase was noted for the sodium / potassium ratio at the high dose level.

A test item-related increase was also noted for the concentration of urea in the blood at the high dose level. Two of 5 individual values at the high dose level were clearly above the highest values of the control group and the remaining 3 values of the high dose group were slightly above or in the range of the highest values of the control group. Due to the increased concentrations of urea in blood at the high dose level, also the ratio of urea in blood (BUN) to the creatinine concentration in the blood was increased, as no changes were noted for the creatinine concentration.

A test item-related decrease was noted at the high dose level for the concentration of cholesterol. Though all individual values of the control group and the high dose group were in the range of background data, 4 of 5 individual high dose values were below the lowest value of the control group.

A statistically significantly increase was noted for aspartate aminotransferase activity (p ≤ 0.01). However, a markedly increased aspartate aminotransferase activity was only noted for one female, whereas the aspartate aminotransferase activities of the other high dosed females were only slightly increased, but within the laboratory background data. However, due to the more pronounced increased aspartate aminotransferase activity that was noted for the male animals, this was considered to be test item-related.

Please refer to section "Overall remarks, attachments".

Endocrine findings:

no effects observed

Urinalysis findings:

not examined

Behaviour (functional findings):

effects observed, treatment-related

Description (incidence and severity):

1) Females:
- 750 mg/kg bw/day: an adverse effect was noted on some of the examined motoric skills (limb rotation, wire maneuver, positional passivity, positive geotropism) for the 4 surviving female animals that were used for the observational screening tests (considered to be test item-related). In addition, a statistically significantly reduced fore- and hindlimb grip strength was noted for the 4 surviving females of the 750 mg test item/kg bw/day dose group (p ≤ 0.01 and p ≤ 0.05), respectively). The grip strength values of the individual animals were below or near the lower range of laboratory background data. The decreased grip strength was considered to be test item-related.

Please refer to section "Overall remarks, attachments".

Immunological findings:

not examined

Organ weight findings including organ / body weight ratios:

effects observed, treatment-related

Histopathological findings: non-neoplastic:

effects observed, treatment-related

Description (incidence and severity):

1) Males:

Adrenals:
A minimal to marked hypertrophy of the cortex was noted for 3 of 5 males of each dose group (two prematurely deceased males of the high dose
group were included).

Heart:
A minimal to moderate necrosis/inflammation was noted for 2 of 5 males of the 75 mg/kg bw/day dose group and 250 mg/kg bw/day dose group, and for 3 of 5 males of the 750/500 mg/kg bw/day dose group (two prematurely deceased males were included).

Kidneys:
Several changes were noted for all examined males of each dose group (basophilic tubules in the cortex, degeneration of the proximal and distal tubules, glomerulonephritis). An increased oil red O staining was noted for 1 of 5 males of the 75 mg/kg bw/day dose group (not considered to be test item related) and for all males of the 750/500 mg/kg bw/day dose group. No increased oil red O staining was noted for the males of the control group and the 250 mg/kg bw/day dose group.

Lungs:
A marked congestion of the lungs was noted for the prematurely deceased male of the 750/500 mg/kg bw/day dose group. A few granulomas were noted for 1 of 5 males of the 250 mg/kg bw/day dose group.

Lymph node (mesenteric):
A slight to minimal sinus histocytosis was noted for 2 of 5 males of the 750/500 mg/kg bw/day dose group (including one prematurely deceased male).

Skeletal muscle:
Minimal to slight muscle degeneration with/without a minimal to slight granulomatous inflammation was noted for 1 of 5 males of the 250 mg/kg bw/day dose group, and for 2 of 5 males of the 750/500 mg/kg bw/day dose group (including one prematurely deceased animal).

Stomach:
A minimal to marked acute inflammation of the mucosa/submucosa was noted for 4 of 5
males of the 75 mg/kg bw/day dose group and 750/500 mg/kg bw/day dose group (including one prematurely deceased male), and for all 5 males of the 250 mg/kg bw/day dose group. Marked to moderate ulceration of the cardia or glandular mucosa was observed for 2 of 5 animals of the 750/500 mg/kg bw/day dose group and a few erosions of the glandular mucosa were noted for one 750/500 mg/kg bw/day dosed male. One male of the 750/500 mg/kg bw/day dose group revealed a moderate squamous cell hyperplasia in the mucosa of the forestomach.

Urinary bladder:
A minimal to moderate hyperplasia of the urothelial cells was noted for 4 of 5 males of the 75 mg/kg bw/day dose group, for all 5 males of the 250 mg/kg bw/day dose group, and for 3 of 5 males of the 750/500 mg/kg bw/day dose group.

Testes:
A microscopic examination was performed on the left and right testis from 8 high dosed males. These 8 left and right testes originated from the 5 animals that were selected for histopathological examination and from 3 further 750/500 mg/kg bw/day dosed males with macroscopic testes findings. From these 8 high dosed animals with microscopic tested examination microscopic testes findings in the form of minimal to slight tubular degeneration / atrophy and moderate to marked tubular dilatation were noted in the testes from 5 animals.
Four of the 5 high dosed males with microscopic testes findings also revealed macroscopic testes findings, which were correlated to the microscopic findings.
In addition, histopathological examination was also performed on the testicles of the 75 and 250 mg/kg bw/day dose group animals. A minimal degeneration/atrophy of the testicles was noted for 1 of 5 animals of the 250 mg/kg bw/day dose group.

2) Females:
Note: all 10 females of the 750 mg/kg bw/day dose group were examined histopathologically: 6 prematurely deceased females and the 4 surviving females. The 4 surviving females were included in the 5 selected 750 mg/kg bw/day dose females that were selected for the histopathological examination at the begin of the study.

Adrenal gland:
A slight hypertrophy of the cortex was noted for 1 of 5 females of the 250 mg/kg bw/day dose
group, and for 2 of 10 females of the 750 mg/kg bw/day dose group (the prematurely deceased
females).

Heart:
A minimal or moderate necrosis/inflammation was noted for 1 of 5 females of the 259 mg/kg bw/day dose group, and for 4 of 10 females of the 750 mg/kg bw/day dose group (all prematurely deceased females).

Kidneys:
For the female animals test item-related kidney changes in the form of basophilic tubules
and degenerated tubules were only noted for one female of the 750 mg/kg bw/day dose group.
An increased oil red O staining was noted for 7 of 10 high dosed females, but also for 4
of 5 females from the control group.

Lungs:
A minimal to moderate congestion of the lungs was noted for 2 of 5 females of the 75 mg/kg bw/day dose group and for 3 of 5 females of the 250 mg/kg bw/day dose group. At the 750 mg/kg bw/day dose level a slight congestion of the lung was noted for one surviving female and a moderate or marked congestion of the lung for two
prematurely deceased animals.
Granulomas were noted for 2 of 10 females of the 750 mg/kg bw/day dose group.

Lymph nodes (mesenteric):
Slight to moderate sinus histocytosis was noted in the mesenteric lymph node from 3 of 5 females of the 75 mg/kg bw/day dose group and 250 mg/kg bw/day dose group, and from 7 of 10 females of the 750 mg/kg bw/day dose group (including four prematurely deceased animals).

Lymph node (cervical):
A marked lymphoid hyperplasia was noted for one prematurely deceased female of the 750 mg/kg bw/day dose group. Furthermore, a marked plasmacytosis was noted for 1/5 female of the 250 mg/kg bw/day dose group (not considered to be test item related) and 2/10 females of the 750 mg/kg bw/day dose group (a moderate plasmacytosis was noted for 3 females of the 750 mg/kg bw/day dose group but also for 2 females of the control group and considered to be
spontaneous).

Skeletal muscle:
Minimal to slight muscle degeneration together with minimal to slight granulomatous inflammation was noted for 5 of 10 female animals (including one prematurely deceased animal) at the 750 mg/kg bw/day dose level.

Spleen:
Minimal to moderate atrophy of the white pulp was noted for 1 of 5 females of the 75 mg/kg bw/day dose group, for 2 of 5 females of the 250 mg/kg bw/day dose group, and for 5 of 10 females of the 750 mg/kg bw/day dose group (including four prematurely deceased females).

Stomach:
A few to several erosions of the glandular mucosa were noted for 5 of 10 females of the 750 mg/kg bw/day dose group (including three prematurely deceased females).
A minimal to slight acute inflammation of the mucosa/submucosa was observed for 3 of
5 females of the 75 mg/kg bw/day dose group and for 2 of 5 females of the 250 mg/kg bw/day
dose group. At the 750 mg/kg bw/day dose level an acute inflammation of the mucosa/submucosa was noted for 9 of 10 females of the 750 mg/kg bw/day dose group (including six prematurely deceased animals).
A marked ulceration of the mucosa was noted for 1 of 10 animals of the 750 mg/kg bw/day dose group (one prematurely deceased animal).
A minimal to moderate squamous cell hyperplasia in the mucosa of the forestomach was noted for 4 of 5 females each of the 75 and 250 mg/kg bw/day dose group and for 1 of 10 females of the 750 mg/kg bw/day dose group.

Thymus:
A severe atrophy was noted for 6 of 10 750 mg/kg bw/day dosed females (six prematurely deceased animals).

Urinary bladder:
A minimal to slight hyperplasia of the urothelial cells was noted for 2 of 5 females of the
75 mg/kg bw/day dose group, for 3 of 5 females of the 250 mg/kg bw/day dose group, and for 3 of 10 animals of the 750 mg/kg bw/day dose group (three prematurely deceased animals).

Uterus:
Changes were noted in the uterus from 3 of 10 females of the 750 mg/kg bw/day dose group in the form of a moderate to marked inflammation and / or a marked haemorrhagic endometrium.

Vagina:
A minimal to moderate mucification of the vaginal mucosa was noted for 2 of 5 animals of the 75 mg/kg bw/day, for 3 of 5 animals of the 250 mg/kg bw/day, and for 4 of 10 animals of the 750 mg/kg bw/day dose group (all prematurely deceased animals).

Please refer to section "Overall remarks, attachments".

Histopathological findings: neoplastic:

not examined

Other effects:

no effects observed

Reproductive function: oestrous cycle:

no effects observed

Reproductive function: sperm measures:

no effects observed

Reproductive performance:

effects observed, treatment-related

Description (incidence and severity):

- 750 mg/kg bw/day: 1/9 pregnant females (animal which died during littering was excluded) delivered only stillbirth, leading to a reduced gestation index of 89 %. Such a single occurrence is in the spontaneous range. However, due to the marked adverse effects that were noted for the prenatal development of the pups at the 750 mg/kg bw/day dose level, the reduced gestation index as well as the delivering of only stillbirths by the one animal has to be considered as test item-related.
Furthermore, a statistically significantly (p ≤ 0.01) elongated gestation length was noted at the 750 mg/kg bw/day dose level. This was considered to be test item-related. In detail, the females showed a gestation length of 23 or 24 test days, whereas in the control group gestation lengths were noted between 21 and 23 test days. The mean gestation length at the 750 mg/kg bw/day dose level was 23.3 test days and above the range of the laboratory background data.

- 750 mg/kg bw/day: the mean number of implantation sites per dam was statistically significantly (p ≤ 0.01) reduced by 25.8% (11.8 implantation sites per dam at the high dose level in comparison to 15.9 implantation sites per dam from the control group). Additionally to the reduced number of implantation sites, an adverse effect of the test item was also noted on the development of the implanted eggs (demonstrated by an increased number of resorptions and a high number of stillbirths). This led to a reduced birth index and a reduced live birth index at the high dose level.
4.9 implantations per dam of the high dose group got lost as a resorption or a stillborn on average, in comparison to only 1.1 lost implantations per dam from the control group. The increased number of resorptions and stillbirths led to a markedly increased post-implantation loss at the high dose level (41.5 % in comparison to only 7.1 % in the control group on the group level).

Please refer to section "Overall remarks, attachments".

CLINICAL SIGNS/DETAILED CLINICAL SIGNS:
Clinical signs:
1) Males:
- 0 mg/kg bw/day: no changes in behaviour, the external appearance or the appearance of the faeces were noted.
- 75 mg/kg bw/day: no changes in behaviour, the external appearance or the faeces were noted that were of toxicological relevance. However, a slight salivation was noted for 2/9 animals on one day each. Such a post-dosing salivation that started briefly after dosing and lasted for a short time is often observed in gavage studies and maybe a reaction to the taste or irritation of the test article, rather than an indication of toxicity
- 250 mg/kg bw/day: salivation was observed in 6/10 animals (not toxicologically relevant). Furthermore, haemorrhagic urine was noted for one animal on 4 consecutive test days. As only 1 animal was affected this observation was considered to be spontaneous.
- 750/500 mg/kg bw/day: salivation was noted for all 8 surviving animals (not toxicologically relevant).
- premature death (75 mg/kg bw/day; one male): the observations that were noted a few days before death during the daily cage side observations (e.g. prone position) were considered to be caused by the misgavage and the following poor health state of the animal.

2) Females:
- 0 mg/kg bw/day: no changes in behaviour, the external appearance or the appearance of the faeces were noted.
- 75 and 250 mg/kg bw/day: no observations were noted that were considered to be of toxicological relevance. In the low dose group, slight to moderate salivation was observed in 2/9 animals during the gestation period. In the 250 mg/kg bw/day dose group, slight to pronounced salivation was observed in 1/10, 7/9 and 4/9 animals during the premating/mating, gestation and lactation period, respectively.
- 750 mg/kg bw/day: salivation was noted in 5/10, 9/10 and 2/4 animals during the premating/mating, gestation and lactation period, respectively (not toxicologically relevant).
- premature deaths (750 mg/kg bw/day): pre-mortal symptoms were noted during the daily cage side observations for all prematurely deceased animals in the form of convulsions, tremors, lateral position, reduced motility, breathing sounds and or a haemorrhagic nose/snout.

Start and duration of observations:
- salivation: with only a few exceptions, salivation started immediately to 5 min after administration and disappeared again between 20 and 60 min after administration.
- reduced motility: a reduced motility was only recorded for 2 animals on their day of death (premature death (one male and one female; high dose group).
For the male animal a reduced motility was soon noted in the morning at work begin on test day 30 and lasted until its premature death on test day 30. Due to the poor health condition of the male no administration of test item was carried out anymore on this day. Hence, no correlation of the symptoms to the time point of administration was possible on this day. In case of the female animal a reduced motility was noted immediately to 5 min after administration in the morning of lactation day 9 and persisted until its premature death during the further course of lactation day 9.
- prone position: prone position was noted for the prematurely deceased male of the 75 mg/kg bw/day dose group on test day 47 and on test day 48. It started between 20 and 60 min after administration on test day 47 and persisted until the premature death on the following day (test day 48).
- convulsions: convulsions were noted for 4 of 6 prematurely deceased high dosed females one day before and/or on their day of death. It started immediately to 2 hours after administration and disappeared between 5 min and 2 hours after administration.
- tremors: tremors were noted for prematurely deceased female of the 750 mg/kg bw/day dose group, on lactation day 1, one day before the animal was found dead on lactation day 2. Tremors were noted immediately to 5 min after administration and disappeared between 20 and 60 min after administration.


Detailed clinical signs:
1) Males:
- 0, 75, 250 and 750/500 mg/kg bw/day: no external observations, no changes in body posture, movement and coordination and in behaviour were noted for the male animals of the treatment groups and the control group.

2) Females:
- 0, 75, 250 and 750 mg/kg bw/day: no external observations, no changes in body posture, movement and coordination and in behaviour were noted for the female animals of the treatment groups and the control group.

MORTALITY:
1) Males:
- 0, 75 and 250 mg/kg bw/day: no test item-related death was noted. One male of the 75 mg/kg bw/day dose group died during test day 48 (two days before
scheduled removal on test day 50). The premature death of the animal was considered to be caused by a misgavage a few days before death and not test item-related.

2) Females:
- 0, 75 and 250 mg/kg bw/day: no test item-related premature death was noted.

BODY WEIGHT AND WEIGHT CHANGES:
1) Males:
- 75 and 250 mg/kg bw/day: no test item-related changes in body weight and body weight gain were noted for the male rats between the control group and the dose groups. Furthermore, no test item-related differences were noted on the body weight at autopsy between the control group and the treatment groups.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related differences in body weight and body weight gain were noted between the female rats of the control group and the female rats of the treatment groups during the premating, the gestation and the lactation period. Furthermore, no test item-related differences were noted on the body weight at autopsy between the control group and the treamtent groups.

Please refer to section "Overall remarks, attachments".

FOOD CONSUMPTION:
1) Males (Pre-mating period):
- 75, 250 and 750/500 mg/kg bw/day: during the pre-mating period (test days 15 to 28) no test item-related difference in food consumption was noted between the rats of the control group and the rats of the treatment groups.

2) Females (Pre-mating, gestation and lactation period):
- 75 and 250 mg/kg bw/day: no test item-related differences in food consumption were noted between the female rats of the control group and the female rats of the treatment groups during the premating, the gestation and the lactation period.
- 750 mg/kg bw/day: no test item-related differences in food consumption were noted during the pre-mating period, the gestation period and the first week of the lactation period (lactation days 1 to 8).

DRINKING WATER CONSUMPTION:
Males and females:
- 75, 250 and 750/500 mg/kg bw/day: no test item-related changes in the consumption of drinking water was noted for the male and female rats.

HAEMATOLOGICAL FINDINGS:
1) Males.
Note: The 5 selected males of the 750/500 mg/kg bw/day dose group that were selected for the laboratory examinations included the 2 prematurely deceased animals).
- 75 mg/kg bw/day: no test item-related influence on the examined haematological parameters were noted.
- 250 and 750/500 mg/kg bw/day: slightly decreased haemoglobin contents were noted (statistically significant at the 250 mg/kg bw/day dose level, p ≤ 0.05). A
comparison with the laboratory background data revealed that 3 of 5 individual values from the males of the intermediate dose group were below the laboratory background range. However, from the high dosed males, the HGB value from only 1 of 5 males was below the laboraotry background range. Hence, as the changes were only slight and showed no dose-response relationship, they were considered to be spontaneous.
- 75 and 750/500 mg/kg bw/day: slightly decreased MCH and MCV values were noted (statistically significant for the MCH value at the 75 mg/kg bw/day dose level (p ≤ 0.05) and for the MCH value and the MCV value at the 750/500 mg/kg bw/day dose level (p ≤ 0.01)). However, no decreased MCH and MCV values were noted at the 250 mg/kg bw/day dose level. All individual values of the control group and the 250 mg/kg b/day dose group are within the laboratory background range for the parameters of MCH and MCV. At the 75 mg/kg bw/day dose level 2 of 5 individual values of the MCH concentration and at the high dose level 3 of 5 individual values each of the MCH concentration and the MCV concentration were below the laboraotry background range.As the MCH and MCV values at the 250 mg/kg bw/day dose level were at the level of the control group, a dose-response relationship from the 75 mg/kg bw/day to the 750/500 mg/kg bw/day dose level was missing. Hence, the changes that were noted for the MCH and MCV values were considered to be spontaneous.

2) Females:
Note: The 5 females of the 750 mg/kg bw/day dose level that were selected for the laboratory examinations included the 4 surviving females and one prematurely deceased female.
- 75 mg/kg bw/day: no test item-related influence on the examined haematological parameters were noted.
- 250 mg/kg bw/day: a slight but statistically significantly decrease of activated partial thromboplastin time (p ≤ 0.01) was noted, whereas the activated partial thromboplastin time at the 750 mg/kg bw/day dose level was again in the range of the control group. As no dose-response relationship was noted, this observation was considered to be spontaneous.

Please refer to section "Overall remarks, attachments".

CLINICAL BIOCHEMISTRY FINDINGS:
1) Males:
- 75 mg/kg bw/day: no test item-related changes for the examined biochemical parameters were noted.
- 250 mg/kg bw/day: the decreased protein (total) concentration that was noted at the intermediate dose group could be considered as test item-related due to its dose-response relationship with the decreased protein (total) concentration that was noted at the high dose level. However, the reduction in the protein (total) concentration that was noted at the intermediate dose level was not considered to be an adverse effect. This was due to the fact that the individual values of the protein (total) concentration at the intermediate dose level were all clearly within the laboratory background data. Furthermore, the decrease was not statistically significant at the intermediate dose level (in contrast to the decreased potassium concentration at the intermediate dose level that was noted for the male animals).

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related changes for the examined biochemical parameters were noted.
- 250 and 750 mg/kg bw/day: the slightly statistically significantly decreased protein (total) and globulin concentrations that were noted at the 250 and the 750 mg/kg bw/day dose group were considered to be spontaneous. All values were in the range of the mean value of the laboratory background data. The significant differences between the control group and the 250 and the 750 mg/kg bw/day dose group may be due to an increased protein (total) concentration at the control group. This assessment is in contrast to those that was made for the male animals for these parameters. However, whereas all protein (total) values from the 750 mg/kg bw/day dosed females were near the mean value of the laboratory background data, a higher variability was noted for the male animals and 2 of 5 values from the 750/500 mg/kg bw/day dosed males were below the lower range of the background data.
- 75, 250 and 750 mg/kg bw/day: the statistically significantly decreased glucose concentrations that were noted were due to an increased glucose concentration at the control group and considered to be spontaneous. All mean values of the treatment groups were at the same level and in the range of the mean value of the laboratory background data.
- 250 and 750 mg/kg bw/day: slightly decreased alanine aminotransferase activities were noted (statistically significant or not). However, increased activities are an indicator for organ damage (alanine aminotransferase release from destroyed cells), whereas decreased activities were not of toxicological relevance and could be considered as spontaneous.

Please refer to section "Overall remarks, attachments".

ENDOCRINE FINDINGS:
Males:
- 75, 250 and 750/500 mg/kg bw/day: no toxicologically relevant changes were noted for the T4 levels of all treatment groups.

NEUROBEHAVIOURAL EXAMINATION:
1) Males:
- 75, 250 and 750/500 mg/kg bw/day: no test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faeces were noted for the male animals of all treatment groups. Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. Also, no test item-related influence on the fore- and hindlimb grip strength was noted for the treated male animals. Lastly, no test item-related differences in spontaneous motility were noted between the control group and the treatment groups. However, statistically significantly reduced numbers of movements were noted at the low dose level (57.1 % below the control value; p ≤ 0.05) and the high dose level (70.2 % below the control value; p ≤ 0.01). These observations were considered to be spontaneous and not test item-related due to the following reasons: a high standard deviation was noted for all groups and nearly all individual values were in the range of laboratory background data (only one animal of the high dose group with only 26 movements in 12 minutes was slightly below the lower laboratory background range).

2) Females:
Note: at the 750 mg/kg bw/day dose level only 4 surviving female animals were still available at the time point of the neurological screening.
- 75 and 250 mg/kg bw/day: no test item-related observations of abnormal behaviour, no adverse effects on motoric skills, changes in the external appearance and the appearance of the faeces were noted. Furthermore, no test item-related differences were noted in body temperature or the hind-leg splay in comparison to the control group. Also, no test item-related influence on the fore- and hindlimb grip strength was noted for the female animals of the low and the intermediate dose group.
- 75 and 750 mg/kg bw/day: the slight but statistically significantly increased hind leg splay that was noted for the animals of the 75 and the 750 mg/kg bw/day dose level (13.4 % and 17.4 % above the control group; p ≤ 0.05) was not considered to be test item related. No dose response relationship was noted and all individual values were in the range of laboratory background data or only slightly above (one animal of the low dose group with a hindleg splay of 10.5 cm).
- 75, 250 and 750 mg/kg bw/day: no test item-related differences in spontaneous motility were noted between the control group and the treatment groups.

Please refer to section "Overall remarks, attachments".

ORGAN WEIGHTS INCLUDING ORGAN/BODY WEIGHT RATIOS:
1) Males:
- 75 mg/kg bw/day: no test item-related changes were noted. The statistically significantly increased relative organ weight of the left kidney (p ≤ 0.05) that was noted revealed no dose-response relationship and was considered to be spontaneous.
- 250 mg/kg bw/day: slight, but statistically significantly decreased organ weights were noted for the absolute organ weights of the left and right epididimydes (p ≤ 0.01 and p ≤ 0.05, respectively). As the changes were only small and no statistically significant changes were noted for the relative organ weights of the left and right epididymis, these changes were considered to be spontaneous.
- 750/500 mg/kg bw/day: more pronounced changes as at the 250 mg/kg bw/day dose level (statistically significant decreased; at p ≤ 0.01) were noted for the absolute organ weights of the left and right epididymis, the combined prostate and seminal vesicle and the liver. However, these changes were considered to be due to the reduced body weight of the 750/500 mg/kg bw/day dosed animals, as no statistically significant changes were noted for the relative organ weights of left and right epididymis, the prostate and seminal vesicle. In case of the liver the relative organ weight was statistically significantly reduced at the high dose level (12.2 % below the control (p < 0.05). However, as the difference to the control for the relative organ weight of the liver was only small and no liver changes were noted during the histopathological examination, this difference was considered to be spontaneous.
- 750/500 mg/kg bw/day: the slight but statistically significantly increased relative organ weight of the brain (p ≤ 0.01) that was noted was also due to the reduced body weight at the dose level, as the absolute organ weight of the brain was in the range of the control group.

2) Females:
- 75 mg/kg bw/day: no test item-related influence on the relative and the absolute organ weights was noted.
- 750 mg/kg bw/day: the statistically significantly decreased absolute organ weights of the heart and the liver (p ≤ 0.05) were considered to be due to the reduced body weight of the 4 surviving female animals, as no statistically significant decrease was noted for the relative organ weights.

Please refer to section "Overall remarks, attachments".

GROSS PATHOLOGICAL FINDINGS:
1) Males:
- 75 and 250 mg/kg bw/day: no test item-related observations were noted for the surviving male animals. However, different observations were noted at the two dose level. These observations were considered to be spontaneous, as they were only noted for 1 (e.g. soft and enlarged left kidney) or 2 animals (observed for 2 animals: an urinary bladder which was filled with content, noted in animals of the low dose group). However, the urinary bladder and the kidneys were considered as target organs during the histopathological examination of the animals of 750/500 dose group.
- premature death (75 mg/kg bw/day; one male): the macroscopic findings at necropsy were considered to be due to the misgavage (abdomen filled with clear liquid or considered to be spontaneous (prostate findings). The observation of soft kidneys was correlated with microscopic kidney findings (e.g. basophilic tubules in the cortex) which were noted for the animal.

2) Females:
- 75 and 250 mg/kg bw/day: no test item-related observations were noted for the female animals. The stomach changes in the form of haemorrhagic foci that were noted for 1 female of the 75 mg/kg bw/day dose group and 1 female animal of the 250 mg/kg bw/day dose group were considered to be spontaneous, as only 1 animal per group was affected. However, due to microscopic and related macroscopic findings at the 750 mg/kg bw/day dose level, the stomach was considered as a target organ.

Please refer to section "Overall remarks, attachments".

HISTOPATHOLOGICAL FINDINGS - NON-NEOPLASTIC
1) Males
Spleen:
A marked atrophy of the white pulp was noted for one male of the 75 mg/kg bw/day dose group.

Thymus:
A severe thymus atrophy was noted for 1 of 5 males each of the 75 mg/kg bw/day dose group and the 750/500 mg/kg bw/day dose group. However, these single occurrences were not considered to be test item-related.
A moderate to marked atrophy was noted for all remaining males of all dose groups. This was considered to be spontaneous, as a marked thymus atrophy was also noted for the male animals of the control group.

2) Females:
Thymus
A moderate to marked atrophy was noted for all remaining females of all dose groups. This was considered to be spontaneous, as a marked thymus atrophy was also noted for the female animals of the control group.

Please refer to section "Overall remarks, attachments".

REPRODUCTIVE FUNCTION- OESTRUS CYCLE
- 75, 250 and 750 mg/kg bw/day: no differences were noted in the mean number of oestrus cycles per dam during the pre-mating and mating period between the female animals of the control group and the female animals of the treatment groups. However, several females with an elongated diestrus stage (between 9 to 16 consecutive test days) were noted before the day of mating. In detail, 2 animals with an elongated diestrus stage were noted in the control group, 4 in the low dose group, 1 in the intermediate dose group and 3 in the high dose group. As 2 animals with an elongated diestrus stage before mating were noted in the control group, the respective findings in the treatment groups were considered as spontaneous.

REPRODUCTIVE FUNCTION - SPERM PARAMETERS
- 75, 250 and 750 mg/kg bw/day: no test item-related influence on spermatogenesis was reported.

REPRODUCTIVE PERFORMANCE:
- 75, 250 and 750 mg/kg bw/day: no test item-related influence on the fertility index of the female rats was noted for any of the treatment groups. All female rats were successfully mated (confirmed by sperm detection). However, 2 female animals of the control group and one female animal of the 75 mg/kg bw/day and the 250 mg/kg bw/day dose group did not become pregnant, leading to a fertility index of 80% for the female animals of the control group and 90% for the animals of the 75 mg/kg bw/day and the 250 mg/kg bw/day dose group. The occurrence of one non-pregnant female each at the low dose level and at the intermediate dose level was considered as spontaneous, as even 2 non-pregnant females were noted at the control group.
- 75 and 250 mg/kg bw/day: no test item-related influence on the gestation index was noted for the female rats.
- 75, 250 and 750 mg/kg bw/day: no test item-related differences were noted in the length of the pre-coital time between the control group and the treatment groups. Conspicuously elongated pre-coital times of 12 test days were noted for one pregnant animal of the 75 mg/kg bw/day dose group and one pregnant animal of the 250 mg/kg bw/day dose group. These single occurrences were considered to be spontaneous. The pre-coital times of the pregnant 750 mg/kg bw/day dosed females (1 to 5 test days) were in the range of the pre-coital times of the pregnant females of the control group (1 to 4 test days).
- 75 and 250 mg/kg bw/day: no test item-related differences were noted for the length of the gestation period between the rats of the control group and the treated rats. The mean gestation length of the animals of the 250 mg/kg bw/day dose group was slightly above the laboratory background range. However, this slight elongation of the gestation length, which was without statistical significance was considered to be spontaneous.
- 75 and 250 mg/kg bw/day: no test item-related differences were noted for the mean number of implantation sites between the control group and dose groups. Furthermore, correlating, the reproductive indices as the birth index, the live birth index and the percentage of post implantation loss revealed no test item-related differences between the control group and the dose groups.

Please refer to section "Overall remakrs, attachments".

Dose descriptor:

LOAEL

Remarks:

(general toxicity)

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Dose descriptor:

NOAEL

Remarks:

(general toxicity)

Effect level:

< 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

haematology

clinical biochemistry

organ weights and organ / body weight ratios

histopathology: non-neoplastic

Critical effects observed:

not specified

Clinical signs:

not examined

Dermal irritation (if dermal study):

not examined

Mortality / viability:

mortality observed, treatment-related

Description (incidence and severity):

Post cull period:
- 75, 250 and 750 mg/kg bw/day: a test item-related decreased viability index was noted for all test item treated groups. In detail, dead pups were noted for 5 of 9 dams of the 75 mg/kg bw/day dose group, 9 of 9 dams of the 250 mg/kg bw/day dose group and for the two remaining dams of the 750 mg/kg bw/day dose group with live pups during the post cull period. In contrast, in the control group dead pups were only noted for 2 of 8 dams.

Please refer to section "Overall remarks, attachments".

Body weight and weight changes:

no effects observed

Food consumption and compound intake (if feeding study):

not examined

Food efficiency:

not examined

Water consumption and compound intake (if drinking water study):

not examined

Ophthalmological findings:

not examined

Haematological findings:

not examined

Clinical biochemistry findings:

not examined

Urinalysis findings:

not examined

Sexual maturation:

not examined

Anogenital distance (AGD):

no effects observed

Nipple retention in male pups:

no effects observed

Organ weight findings including organ / body weight ratios:

no effects observed

Gross pathological findings:

no effects observed

Histopathological findings:

no effects observed

Other effects:

no effects observed

Behaviour (functional findings):

not examined

Developmental immunotoxicity:

not examined

MORTALITY / VIABILITY:
- 75 and 250 mg/kg bw/day: no test item-related differences were noted for the mean number of pups born (alive and dead) and the mean number of live born pups between the control group and the dose groups.
Pre-culled period:
- 75 and 250 mg/kg bw/day: no difference between the control group and the two treatment groups was noted for the viability index between lactation days 1 and 4.
- 750 mg/kg bw/day: most of the live born pups died between lactation days 0/1 and 4 (46 prematurely deceased pups of 62 live born pups). The high mortality of the pups was due to bad health conditions of the dams (demonstrated by the premature deaths of altogether 6 dams at the begin or during the lactation period) and bad health conditions of the pups (demonstrated by a reduced body weight on lactation day 1). The overview of the litter fate is as follows:
- 2 of 10 pregnant females did not deliver live pups (one female only delivered stillbirths and another female died during littering).
- 8 of 10 pregnant females delivered live pups.
- 5 of 8 females with live pups showed a total litter loss during the pre-cull period. Three of these 5 females died a few days after the death of all their pups.
- 1 female was found dead (on lactation day 2) and the pups were prematurely sacrificed.
- 1 female lost all pups during the pre- and the post cull period.
- 1 female lost no pups during the pre-cull period but most of the pups during the post-cull period. However, the female was the only female with surviving pups at the end of the lactation period.

BODY WEIGHTS AND WEIGHT CHANGES:
1) Pups:
- 75 and 250 mg/kg bw/day: no test item-related difference was noted between the mean body weight of the pups from the dams of the control group and the mean body weight of the pups from the dams of the 75 mg/kg bw/day and the 250 mg/kg bw/day dose group on lactation days 1, 4 and 13. The reduction in pup body weight in comparison to the control group that was noted at the 250 mg/kg bw/day dose level on lactation day 4 (13.5 % below the value of the control group; males and females combined) was considered to be spontaneous as it was only slight and statistically not significant.
- 750 mg/kg bw/day: a statistically significantly and markedly reduced pup body weight was noted on lactation day 1 (p ≤ 0.01). The difference in pup body weight between the pups of treatment group and the pups of the control group increased during the further course of the lactation period. However, only a few pups of the dose group were still available during the further course of the lactation period (16 available pups on lactation day 4 and 3 available pups on lactation day 13).
- 75, 250 and 750 mg/kg bw/day: no runt was noted in the control group and in the treatment groups.

2) Litter:
- 75 and 250 mg/kg bw/day: statistically significantly (p ≤ 0.05 / 0.01) reduced litter weights were noted on lactation day 13 (male and female pups combined), due to the high number of prematurely deceased pups between lactation day 4 and 13.
- 750 mg/kg bw/day: the litter weight (male and female pups combined) was reduced on lactation days 1, 4 and 13 (on lactation day 1: statistically significant at p ≤ 0.01; on lactation day 4 and 13: inappropriate for statistics) due to the reduced number of live born pups and the reduced pup body weights. However, at the dose level only 2 litters were still available on lactation day 4 and only 1 on lactation day 13.

GROSS PATHOLOGICAL FINDINGS:
- 75, 250 and 750 mg/kg bw/day: no gross abnormalities (e.g. malformations or variations) were noted during the macroscopic external examination of the control pups and the pups from the dams treated with the test item after terminal sacrifice on lactation day 13 or for the pups that died during the lactation period .

ORGAN WEIGHT FINDINGS INCLUDING ORGAN/BODY WEIGHT RATIO.
- 75 and 250 mg/kg bw/day: the weight of the thyroid glands of the pups revealed no test item-related differences between the control group and the pups of the treatment groups.
- 750 mg/kg bw/day: only the values from the pups of one dam were available. They were 19.0 % (left thyroids of the pups) or 42.5 % (right thyroids of the pups) below the values of the control group. This maybe due to the reduced body weight of both pups. However, due to the low number of available pups at the high dose level, an assessment about an effect on the thyroid weight is not possible.

HISTOPATHOLOGICAL FINDINGS
- 75 and 250 mg/kg bw/day: no test item-related changes were noted for the thyroid glands from the pups of the control group and the pups from the treatment groups.
- 750 mg/kg bw/day: thyroid glands from the 2 pups of one dam revealed no test item-related changes.

MALE TO FEMALE RATIO OF THE PUPS:
- 75, 250 and 750 mg/kg bw: no test item-related influence on the male to female ratio was noted for all treatment groups. However, male to female ratios that markedly differed from the expected value (around 1.0) were noted at the 75 mg/kg bw/day and the 750 mg/kg bw/day dose group with opposite directions (increase and decrease value, respectively). However, the values of the male to female ratios that were noted for the 75 mg/kg bw/day and the 750 mg/kg bw/day dose group were within (low dose group) or only slightly below (high dose group) the range of laboratory background data. Furthermore, no consistent trend was noted. Whereas at the 75 mg/kg bw/day dose level more male pups as female pups were noted, the opposite result was noted at the 750 mg/kg bw/day dose level. Hence, the differences from the expected ratio were considered to be spontaneous.

NUMBER OF LIVE PUPS:
- 75 and 250 mg/kg bw/day: statistically significantly (p ≤ 0.05 / 0.01) reduced numbers of live pups (male and female pups combined) in comparison to the control group were noted on lactation day 13 at the 75 mg/kg bw/day and the 250 mg/kg bw/day dose level. This was due to the high number of prematurely deceased pups between lactation day 4 and 13.
- 750 mg/kg bw/day: statistically significantly reduced numbers of live pups (male and female pups combined) were already noted on lactation day 1 (p ≤ 0.01), due to the reduced number of live born pups.

ANO-GENITAL DISTANCE:
- 75, 250, and 750 mg/kg bw/day: no test item-related influence was noted on the absolute and the relative ano-genital distance of the male and the female pups of all treatment groups.
However, whereas no statistically significant differences between the control group and the treatment groups were noted for the absolute ano-genital distance, statistically significantly increased values were noted for the relative ano-genital distance of the male pups.
In detail, the relative ano-genital distance of the male pups was 12.0 % above the value of the control group (p ≤ 0.05) at the 250 mg/kg bw/day dose level and 27.2 % above the value of the control group (p ≤ 0.01) at the 750 mg/kg bw/day dose level 8inappropriate for statistis due to the low number of only 2 dams with surviving pups on lactation day 4).
Similarly increased values were noted for the relative ano-genital distance of the female pups of the 250 mg/kg bw/day and the 750 mg/kg bw/day dose group (10.9 % or 25.5 % above the value of the control group), but they failed statistical significance.
The differences that were noted at the 250 mg/kg bw/day and the750 mg/kg bw/day dose level for the relative ano-genital distance were due to a reduced pup body weight that was noted at the 250 mg/kg bw/day (considered as spontaneously) and the 750 mg/kg bw/day dose level (considered as test item-related). Hence, the increased ano-genital distance at the 250 mg/kg bw/day and the 750 mg/kg bw/day dose level was a secondary effect of the reduced pup body weight and not a test item-related effect on genital development. Furthermore, all absolute values of the ano-genital distance from the animals of the 250 mg/kg bw/day and the 750 mg/kg bw/day dose group were inside the range of the laboratory background data.

EXAMINATION OF THE MALE PUPS FOR NIPPLES:
- 75, 250 and 750 mg/kg bw/day: no test item-related difference in the number of nipples was noted between the male pups of the control group and in the male pups of the treatment groups.

T4 HORMONE DETERMINATION:
- 75 and 250 mg/kg bw/day: no test item-related changes were noted for the T4 levels of the male and female pups.
- 750 mg/kg bw/day: T4 levels of one examined male and one examined female pup from one dam were available, which were markedly below the control group. However, an assessment about an effect on the T4 level is not possible with such a small number of examined pups.

Dose descriptor:

LOAEL

Generation:

F1

Effect level:

75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Dose descriptor:

NOAEL

Generation:

F1

Effect level:

< 75 mg/kg bw/day (actual dose received)

Based on:

test mat.

Sex:

male/female

Basis for effect level:

viability

Critical effects observed:

not specified

Reproductive effects observed:

yes

Lowest effective dose / conc.:

250 mg/kg bw/day (actual dose received)

Treatment related:

yes

Relation to other toxic effects:

reproductive effects as a secondary non-specific consequence of other toxic effects

Conclusions:

In the current combined repeated dose toxicity study with the reproduction/developmental toxicity screening test, cesium fluoroaluminate in water was administered via gavage to groups of male and female rats (n = 10 animals/sex/group) at dose levels of 75, 250 and 750 mg/kg bw/day (from test day 38 onward 500 mg/kg bw/day as high dose level for males only due to mortality). The administration occurred once daily for males during 2 weeks prior to mating, mating period (14 days at maximum) and post-mating period until one day before sacrifice (total treatment days: 35 days) and for females during 2 weeks prior to mating, mating period (14 days at maximum) and lactation period until lactation day 13 (total treatment days: 50 to 62 days). A vehicle control group was run concurrently.

During the observation of the parental animals, no test item-related effects were observed for water consumption and T4 hormone levels (males only). Mortality was observed in males of the 750 mg/kg bw/day dose group. Two of 10 males died on test days 30 and 36 prior to the reduction of the dose level to 500 mg/kg bw/day. In addition, 6/10 females of the 750 mg/kg bw/day dose level died during the lactation period (between lactation days 1 and 9). Furthermore, test item related clinical signs were noted for males (haemorrhagic nose/snout; n = 3/8 animals) and females (piloerection; n = 4/10 animals (surviving animals)) at the high dose level. The neurological screening revealed test item-related effects for the surviving females that were dosed with 750 mg/kg bw/day in the form of limited motoric skills and a reduced fore- and hindlimb grip strength.
Body weight was reduced for the male animals that were dosed with 750/500 mg/kg bw/day and for the female animals that were dosed with 750 mg/kg bw/day, which was considered to be test item-related. In addition, the four surviving female animals showed a reduced food consumption during lactation days 8 to 12, which maybe due to damaged teeth and/or poor health conditions.
Test item related effects were also observed during the haematological investigation. At the 250 mg/kg bw/day dose level, test item related effects were observed for white blood cells of males. Regarding the subunits of the white blood cells, statistically significant increases were noted for the numbers of neutrophilic and eosinophilic granulocytes and these findings were considered to be test item-related, as more pronounced increases were noted at the high dose level (dose-response relationship). Some value were above the background data of the laboratory. At the 750/500 mg test item/kg bw/day dose level the increase in the number of white blood cells of males was more pronounced as at the intermediate dose level and statistically significant. The cell numbers of nearly all subunits of the white blood cells (cell numbers of lymphocytes, monocytes, neutrophilic, eosinophilic and basophilic granulocytes, with the exception of the small population of large unstained cells) were markedly increased (statistically significant or not). All changes that were noted at the high dose level for the above-mentioned parameters were considered to be test item-related.

Furthermore, increased numbers of neutrophilic granulocytes, eosinophilic granulocytes and monocytes were noted in females at the intermediate and the high dose level (statistically significant or not statistically significant), which were considered to be test item-related. The number of neutrophilic granulocytes showed a clear dose response-relationship. Some of the data were above the background data of the laboratory. A dose response-relationship was also noted for the increase in the number of monocytes from the 250 mg/kg bw/day dose group to the 750 mg/kg bw/day dose group. With one exception, all individual values from the animals of the intermediate and the high dose group were above the individual values from the control group and in the upper range of the background data. In case of the number of eosinophilic granulocytes all individual values from the females of the 250 mg/kg bw/day dose group (5 of 5) and 3 of 5 individual values from the females of the 750 mg/kg bw/day dose group were above the values from the control group and above the background range. Though a dose response-relationship was missing, the distinctly increased numbers of eosinophilic granulocytes in comparison to the control group that were noted for altogether 8 of 10 females of the intermediate and the high dose group were considered to be test item-related.

In addition to the test item-related haematological findings, test item-related effects were observed in males during the biochemical investigation. At the 250 mg/kg bw/day dose level, a slight but statistically significantly decreased potassium concentration was noted. This small reduction was considered to be test item-related due to the test item-related reductions that were noted for the potassium concentrations at the 750/500 mg/kg bw/day dose level of the male and more pronounced of the female animals. At 750/500 mg test item/kg bw/day, test item-related changes were noted for the potassium concentration, the aspartate aminotransferase activity, the protein (total), the albumin and globulin concentration and the urea in blood concentration. In females, test item-related changes were noted for the electrolyte levels (potassium, chloride and calcium concentration), the urea in blood concentration, the cholesterol concentration and the aspartate aminotransferase activity at the 750 mg/kg bw/day dose level.

Macroscopical and microscopical findings that were considered to be caused by the test item were recorded for male and female parental animals. At the 750/500 mg/kg bw/day dose level, macroscopical findings were noted in form of enlarged, small or malpositioned testes and absolute and relative testes weights were noted to be increased in males. Histopathologically, minimal to slight tubular degeneration/atrophy and moderate to marked tubular dilatation were observed in testes and correlated with the macroscopical findings. A minimal degeneration/atrophy of testicles was noted in one animal of the 250 mg/kg bw/day dose group. In addition, adverse effects on thymus weight (decreased absolute and relative weight) were noted for males at 250 and 750/500 mg/kg bw/day dose level, which is in accordance with the observation of thymus atrophy that was noted during histopathology. The four surviving female animals of the 750 mg/kg bw/day dose group revealed pale and broken teeth. The effect on teeth was mostly due to high amount of fluoride in the test item. Moreover, the female animals revealed decreased absolute and relative thymus weight at 250 and 750 mg/kg bw/day. A severe atrophy of thymus was noted in 6/10 females of the high dose group, therefore, the reduced thymus weight was in consistency with histopathological findings and was considered to be test item-related.

Besides the above mentioned histopathological findings, test item related effects were made for adrenals (minimal to marked hypertrophy of the cortex at all dose groups), heart (minimal to moderate necrosis/inflammation at all dose groups), kidneys (basophilic tubules in the cortex, degeneration of the proximal and distal tubules, glomerulonephritis at all dose levels), lungs (marked congestion at high dose level), mesenteric lymph node (slight to minimal sinus histiocytosis at high dose level), skeletal muscle (minimal to slight muscle degeneration with/without minimal to slight granulomatous inflammation at intermediate and high dose levels), stomach (minimal to marked acute inflammation of the mucosa/submucosa at all dose levels; marked to moderate ulceration of the cardia or glandular mucosa at high dose level; few erosions of the glandular mucosa at high dose level; moderate squamous cell hyperplasia in mucosa of the forestomach at high dose level), urinary bladder (minimal to moderate hyperplasia of the urothelial cells at all dose levels) in parental male animals. In addition, in parental female animals test item-related effects were noted for adrenal gland (slight hypertrophy of the cortex at intermediate and high dose levels), heart ( minimal to moderate necrosis/inflammation at intermediate and high dose levels), kidney (basophilic tubules and degenerated tubules in one female at the high dose level), lungs (slight to moderate congestion at all dose levels) mesenteric lymph nodes (slight to moderate sinus histiocytosis at all dose levels), cervical lymph nodes (marked lymphoid hyperplasia at high dose level), skeletal muscle (minimal to slight muscle degeneration with minimal to slight granulomatous inflammation at the high dose level), spleen (minimal to moderate atrophy of the white pulp at all dose level), stomach (minimal to slight acute inflammation of the mucosa/submucosa at all dose levels; marked ulceration of the mucosa in one animal of the high dose group; minimal to moderate squamous cell hyperplasia in the mucosa of the forestomach at all dose levels; a few to several erosions of the glandular mucosa at the high dose level), thymus (severe atrophy at the high dose level), urinary bladder (minimal to slight hyperplasia of the urothelial cells at all dose levels), uterus (moderate to marked inflammation and/or marked haemorrhagic endometrium at the high dose level) and vagina (minimal to moderate mucification of the vaginal mucosa at all dose levels).

Regarding reproduction toxicity, fertility of male and female animals is unaffected during the course of this study. An elongated gestation length and a reduced gestation index was noted for the female animals at 750 mg test item/kg bw/day. Furthermore, the number of implantation sites per dam was reduced and the number of resorptions and stillbirths was increased at the high dose level. The increased number of resorptions and stillbirths led to a reduced birth index, a reduced live birth index and a markedly increased post-implantation loss at the 750 mg/kg bw/day dose level. However, these findings are attributed to the overt toxicity with mortality (6 out of 10 animals died) and should therefore not be considered as primariy reprotoxic effect.

Lastly, no test item-related effects were observed on the offspring of the dosed parental animals for body weight, sex ratio, number of live pups, ano-genital distance, nipple retention (male pups only), organ weights, gross pathology and histopathology. A test item-related decreased viability index was noted at all dose levels.

In conclusion, the no observed adverse effect level (NOAEL) for systemic toxicity in parental animals is below 75 mg/kg bw/day based on haematology, clinical chemistry, organ weights and histopathology.

Furthermore, fertility of male and female animals was unaffected during the course of this study. Adverse effects were observed for gestation length, gestation index, number of implantation sites, number of resorptions and stillbirths at the 750 mg/kg bw/day dose level. However, the adverse findings are attributed to the overt toxicity with mortality (6 out of 10 females died) and should therefore not be considered as primariy reprotoxic effect. Therefore, cesium fluoroaluminate is not considered to be a primary reprotoxic substance, since adverse effects on the fertility were only seen at signs of excessive toxicity, involving death of 6 of 10 female and 2 of 10 males. Consequently, the criterial for classification and labelling as reprotoxic (impairment of fertility) are not met.

No adverse effect was noted on the development of the pups at any dose levels. At 75 and 250 mg test item/kg bw/day a reduced viability index was observed during the post-cull period. At the maternal lethal dose of 750 mg test item/kg bw/day an adverse effect was also noted in form of a reduced viability index during the pre- and post-cull period. Therefore, cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling.

Overall, a LOAEL of 75 mg/kg bw/day is derived for cesium fluoroaluminate based on a decreased pup viability index during the post-cull period.

Effect on fertility: via oral route

Study duration:

subacute

Species:

rat

Effect on fertility: via inhalation route

Endpoint conclusion:

no study available

Effect on fertility: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422, 2016), cesium fluoroaluminate in water was administered via gavage to groups of male and female rats (n = 10 animals/sex/group) at dose levels of 75, 250 and 750 mg/kg bw/day (from test day 38 onward 500 mg/kg bw/day as high dose level for males only due to mortality). The administration occurred once daily for males during 2 weeks prior to mating, mating period (14 days at maximum) and post-mating period until one day before sacrifice (total treatment days: 35 days) and for females during 2 weeks prior to mating, mating period (14 days at maximum) and lactation period until lactation day 13 (total treatment days: 50 to 62 days). A vehicle control group was run concurrently.

Fertility of male and female animals is unaffected during the course of this study. An elongated gestation length and a reduced gestation index was noted for the female animals at 750 mg test item/kg bw/day. Furthermore, the number of implantation sites per dam was reduced and the number of resorptions and stillbirths was increased at the high dose level. The increased number of resorptions and stillbirths led to a reduced birth index, a reduced live birth index and a markedly increased post-implantation loss at the 750 mg/kg bw/day dose level. However, these findings are attributed to the excessive toxicity with mortality (6 out of 10 females and 2 of 10 males died) and should therefore not be considered as primariy reprotoxic effect. Consequently, the criterial for classification and labelling as reprotoxic (impairment of fertility) are not met.

Effects on developmental toxicity

Description of key information

A combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422 (2016); GLP) with cesium fluoroaluminate was conducted in rats. No adverse effect was noted on the development of the pups at any dose levels. At 75 and 250 mg test item/kg bw/day a reduced viability index was observed during the post-cull period. At the maternal lethal dose of 750 mg test item/kg bw/day an adverse effect was also noted in form of a reduced viability index during the pre- and post-cull period. Therefore, cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling.

Effect on developmental toxicity: via oral route

Endpoint conclusion:

adverse effect observed

Dose descriptor:

LOAEL

75 mg/kg bw/day

Study duration:

subacute

Species:

rat

Effect on developmental toxicity: via inhalation route

Endpoint conclusion:

no study available

Effect on developmental toxicity: via dermal route

Endpoint conclusion:

no study available

Additional information

In a combined repeated dose toxicity study with the reproduction/developmental toxicity screening test (OECD 422, 2016), cesium fluoroaluminate in water was administered via gavage to groups of male and female rats (n = 10 animals/sex/group) at dose levels of 75, 250 and 750 mg/kg bw/day (from test day 38 onward 500 mg/kg bw/day as high dose level for males only due to mortality). The administration occurred once daily for males during 2 weeks prior to mating, mating period (14 days at maximum) and post-mating period until one day before sacrifice (total treatment days: 35 days) and for females during 2 weeks prior to mating, mating period (14 days at maximum) and lactation period until lactation day 13 (total treatment days: 50 to 62 days). A vehicle control group was run concurrently. No test item-related effects were observed on the offspring of the dosed parental animals for body weight, sex ratio, number of live pups, ano-genital distance, nipple retention (male pups only), organ weights, gross pathology and histopathology.

At 75 and 250 mg test item/kg bw/day a reduced viability index was observed during the post-cull period. At the maternal lethal dose of 750 mg test item/kg bw/day an adverse effect was also noted in form of a reduced viability index during the pre- and post-cull period. Therefore, cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling.

Overall, a LOAEL of 75 mg/kg bw/day is derived for cesium fluoroaluminate based on a decreased pup viability index during the post-cull period.

Justification for classification or non-classification

Cesium fluoroaluminate is not considered to be a primary reprotoxic substance, since adverse effects on the fertility were only seen at signs of excessive toxicity, involving death of 6 of 10 female and 2 of 10 animals. The criteria for classification and labelling in accordance with regulation EC 1272/2008 are not met, hence no classification as reprotoxic (fertility impairment) required.

Cesium fluoroaluminate caused a dose-dependent decrease of the viability index during the post-cull period, starting at the lowest dose administered. Due to the unclear etiology (either being a direct developmental toxicity or an increased sensitivity of the newborn being exposed via lactation), further studies are required before a definitive decision can be made with regard to classification and labelling for the endpoint reproductive toxicity (developmental toxicity).